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Serum Response Factor Modulates Neuron Survival During Peripheral Axon Injury Sina Stern1,3, Daniela Sinske1,2 and Bernd Knöll1,2*

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Serum Response Factor Modulates Neuron Survival During Peripheral Axon Injury Sina Stern1,3, Daniela Sinske1,2 and Bernd Knöll1,2* Stern et al. Journal of Neuroinflammation 2012, 9:78 JOURNAL OF http://www.jneuroinflammation.com/content/9/1/78 NEUROINFLAMMATION RESEARCH Open Access Serum response factor modulates neuron survival during peripheral axon injury Sina Stern1,3, Daniela Sinske1,2 and Bernd Knöll1,2* Abstract Background: The transcription factor SRF (serum response factor) mediates neuronal survival in vitro. However, data available so far suggest that SRF is largely dispensable for neuron survival during physiological brain function. Findings: Here, we demonstrate that upon neuronal injury, that is facial nerve transection, constitutively-active SRF- VP16 enhances motorneuron survival. SRF-VP16 suppressed active caspase 3 abundance in vitro and enhanced neuron survival upon camptothecin induced apoptosis. Following nerve fiber injury in vitro, SRF-VP16 improved survival of neurons and re-growth of severed neurites. Further, SRF-VP16 enhanced immune responses (that is microglia and T cell activation) associated with neuronal injury in vivo. Genome-wide transcriptomics identified target genes associated with axonal injury and modulated by SRF-VP16. Conclusion: In sum, this is a first report describing a neuronal injury-related survival function for SRF. Keywords: Facial nerve, Immune cell, Motorneuron, Regeneration, SRF, Axon, Microglia Background as SRF target gene in the same study [5]. In primary cor- The gene regulator SRF modulates multiple aspects of tical neurons, SRF overexpression mediates BDNF- neuronal motility. In SRF-deficient mice, cell migration, dependent cell survival in various paradigms of neuronal neurite outgrowth, branching, growth cone shape and axon injury [6]. Also, SRF conveys expression of the immedi- guidance are impaired. In turn, constitutively-active SRF- ate early gene (IEG) Cyr61 during neuronal cell death VP16, a fusion protein of SRF and the viral VP16 transacti- [7]. vation domain, enhances neuronal motility [1]. Thus, SRF’s SRF operates through interaction with co-factors of impact on physiological neuronal motility might proof the MRTF (myocardin-related transcription factors) and beneficial also during axonal regeneration, that is the stimu- TCF (ternary complex factors) family. Through inter- lation of regrowth of severed nerve fibers. action with TCFs SRF can mediate an IEG response of In addition to cell differentiation, SRF has been impli- for example c-fos, Egr1 and Arc. IEGs are well-estab- cated in cell survival of various cell types including lished molecular switches of cell survival vs. cell death hepatocytes [2], thymocytes [3], heart cells [4], and dur- [8]. Further, while interacting with MRTFs SRF directs ing embryogenesis [5]. In embryonic stem cells lacking expression of actin isoforms (Acta, Actb, Actc) or actin- SRF apoptosis was strongly upregulated [4]. The latter binding proteins (for example tropomyosin, calponin and result is in line with downregulation of the antiapoptotic gelsolin) thereby regulating cytoskeletal dynamics [1,9]. protein Bcl-2 upon SRF-deficiency. Bcl-2 was identified Similar to SRF, MRTF-A and the TCF Elk-1 enhance cell survival of primary neurons [6,10–13] and non-neuronal cells [14]. In opposite to primary neurons, cell survival and * Correspondence: [email protected] apoptosis are not overtly altered during physiological ner- 1 Department Molecular Biology, Interfaculty Institute for Cell Biology, vous system development as revealed by SRF-deficient mice Eberhard Karls University Tübingen, Auf der Morgenstelle 15, Tübingen, – 72076, Germany [15 17]. Indeed, apoptosis was only elevated in the subven- 2Current addresses: Institute for Physiological Chemistry, Ulm University, Ulm, tricular zone of SRF-deficient mice [15] but not documen- 89081, Germany ted in for example cortical, hippocampal, striatal and Full list of author information is available at the end of the article © 2012 Stern et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Stern et al. Journal of Neuroinflammation 2012, 9:78 Page 2 of 12 http://www.jneuroinflammation.com/content/9/1/78 peripheral neurons [15–17]. This suggests that SRF is not a antibodies (1:500; Vector) and peroxidase-based detection major neuronal survival regulator during physiological systems using the ABC complex (Vector) and DAB as sub- brain development. strate. Primary antibodies included anti-IBA1 (rabbit, 1:500; As mentioned above, SRF and co-factors mediate in- Wako) and anti-CD3 (mouse, 1:1,000; Dr. G. Jung, jury-related neuronal survival in vitro. Thus, an in vivo Tübingen University). function of SRF in neuronal survival (which has not been demonstrated so far) might become apparent dur- Cell biology ing application of neuronal injury. Primary neurons were prepared as before [20]. Hippocam- Here we applied facial nerve injury in adult mice to inves- pal neurons derived from wild-type or SRF-deficient mice tigate a role of SRF-VP16 in survival of facial motorneurons [15] were electroporated with SRF-VP16 or SRF-ΔMADS- in vivo. In mice, the bilateral facial nerve innervates muscles VP16 and cultured for 72 h. Neurons were electroporated regulating whisker pad and eyelid movements, for example with 3 μg of the plasmids using Amaxa nucleofection [18]. Facial nerve axotomy is a model system for studying resulting on average in 30% to 40% transfected cells. Neu- motorneuron survival, axonal regeneration as well as rons were stimulated for 1 h with myelin (12 μg/ml). Pro- neuron and immune cell interactions during neuronal in- tein lysates were prepared as before [21]. Rabbit anti-active jury. We observed an SRF-VP16 dependent increase in caspase 3 (Cell Signaling; 1:1,000) and mouse anti-GAPDH motorneuron survival in vivo. In addition, SRF-VP16 (Acris; 1:50,000) antibodies were used. enhanced outgrowth and survival of transected primary For neuronal injury experiments in vitro, hippocampal neurons in vitro. Mechanistically this SRF-VP16 function neurons were grown on poly-L-lysine and laminin coated involves suppression of active caspase 3 expression in vitro video dishes. One neurite/neuron was cut with a micro- and increased microglia and T cell activation around trans- scalpel driven by an InjectMan® NI 2 Micromanipulator ected motorneurons in vivo. Finally, using transcriptomics, (Eppendorf). The cell reaction was monitored in a life cell we provide axonal injury-induced and SRF-VP16 modu- imaging set-up (37°C, 5% CO2; Zeiss, Axiovert 200 M) lated target genes potentially associated with neuronal every 5 min for a total of 6 h. Ten neurons/condition in 13 survival. independent experiments were evaluated. Neurons were infected with 1 × 108 PFU/ml adenoviral Methods particles expressing GFP alone, SRF-ΔMADS-VP16:GFP Facial nerve transection or SRF-VP16:GFP 5 h after plating. The next day, cul- The facial nerve transection was performed as described in tures were treated overnight (17 h) with camptothecin at [19]. Adult wild-type mice (>2 month) were anaesthetized, 0.1, 1, or 3 μΜ followed by immunocytochemistry. a skin incision was made behind the left ear and the facial nerve was exposed. In experiments with no virus applica- Immunocytochemistry tion, the nerve was transected with small microscissors Cells were fixed for 15 min in 4% PFA/5% Sucrose/PBS, about 2 mm posterior to the foramen stylomastoideum. permeabilized for 5 min in 0.1% Triton-X-100/PBS and For viral infection, 1 μl virus was injected into the facial blocked for 30 min in 2% BSA/PBS. Primary antibodies nerveusinga26GHamiltonsyringe.Afterwards,thenerve were incubated for 2 h at room temperature as follows: was transected and another 1 μl of virus was injected into rabbit anti-active caspase 3 (Cell Signaling; 1:750; the nerve stump. Of note, this virus injection with a syringe #6991), mouse anti-GFP (Roche; 1:1,000). First anti- causes already a facial nerve lesion. Therefore it is only pos- bodies were detected with Alexa 488, or 546 conjugated sibletodelineateSRF-VP16specificeffectsonthebasisof secondary antibodies (1:1,000; Molecular Probes), fol- experiments employing control virus, SRF-ΔMADS-VP16. lowed by DAPI-staining. Cesium-chloride purified SRF-VP16 (4.6 × 1012 PFU/mL) and SRF-ΔMADS-VP16 (4.9 × 1012 PFU/mL) adenoviral Microarrays particles were purchased from Vector Biolabs. Both viruses The facial nuclei were dissected from 300 μmbrainstem drive GFP expression via a second CMV promoter. Ab- sections prepared with a tissue chopper using tungsten nee- sence of eyelid closure and whisker movement ensured suc- dles. Facial nuclei of four mice/ condition were pooled and cessful nerve transection. All experiments are in resulted on average between 0.5 and 1 μgRNA.TotalRNA accordance with institutional regulations by the local ani- was isolated with the RNeasy kit (Qiagen). RNA of 0.1 μg mal ethical committee (Regierungspräsidium Tübingen). was processed on Affymetrix GeneChips (Mouse Gene 1.0 ST array) according to protocols of the Microarray Facility Histology Tübingen (http://www.microarray-facility.com/cms/index. Brains were fixed in 4% PFA/PBS overnight followed by php). Raw data normalized to the control sample were ana- preparation of 60 μm vibratome slices. Immunohistochem- lyzed in such way that only genes with a fold-change of ≥ istry was performed using Biotin-conjugated secondary 1.5 (up- or down-regulated) were carried forward. Genes Stern et al. Journal of Neuroinflammation 2012, 9:78 Page 3 of 12 http://www.jneuroinflammation.com/content/9/1/78 were considered SRF-VP16 specific if their fold-change dif- injury, that is unilateral de-afferentiation of facial motor- fered two-fold from the respective factor obtained for SRF- neurons in mice (Figure 1A). The transected facial nerve ΔMADS-VP16. was infected with viral particles expressing GFP in addition to SRF-ΔMADS-VP16 or SRF-VP16. SRF-VP16 consists of Quantitative real-time PCR (qPCR) SRF fused to the viral VP16 transactivation domain.
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